The major objectives of this grant proposal are: to assess the effect of acute and chronic diabetes on myocardial intermediary energy metabolism; and to elucidate possible mechanisms responsible for the impairment of substrate utilization and its contribution to diabetic cardiomyopathy. The experimental model will be freshly isolated calcium-tolerant heart myocytes from acutely and chronically streptozotocin-diabetic rats. A secondary objective is to culture these isolated cells in a serum-free, chemically defined medium for the study of hormonal interaction on myocyte metabolism. Freshly isolated cardiac cells represent a fairly new model for studying myocardial metabolism. Despite the fact that they are quiescent, we previously reported that alterations in substrate utilization found in isolated perfused hearts from diabetic rats are also present in isolated myocytes. Preliminary studies from this laboratory have further demonstrated significant differences in substrate utilization patterns in freshly isolated myocytes from acutely and chronically diabetic rats. These may relate to differences in their in vivo diabetic state. In addition, the altered substrate metabolism in chronic diabetes may lead to an energy deficient state. It is therefore important to examine in the isolated myocytes mechanisms that may result in defective energy metabolism. These include determinations of oxidation of selected exogenous substrates, substrate flux through major metabolic pathways, endogenous substrate turnover and availability as energy sources, marker enzyme activities, pertinent intermediary metabolite and energy-rich phosphate levels under conditions resembling those found in normal and diabetic animals. In order to better understand hormonal influences on substrate metabolism, the isolated myocytes will be cultured in a serum-free, hormone-supplemented medium and the metabolic fate of selected substrates will be examined.